Xenotropic Murine Leukemia Virus Related Virus (XMRV) was first identified in prostate tissues from prostate cancer patients. Subsequent studies on the prevalence of the virus have produced inconsistent results, leaving the link between the virus and prostate cancer unclear. We applied a quantitative real-time PCR assay and two well-controlled immunohistochemistry (IHC) assays for the detection of XMRV in freshly obtained and archival tissue specimens. Our real-time PCR assay reliably detected XMRV DNA from a single cells worth of genomic DNA (gDNA) from 22Rv1 cells, an XMRV-infected cell line, even in the presence of a vast excess of uninfected cell gDNA. Our IHC assays utilized two rabbit polyclonal antisera specific for murine leukemia virus, p30CA and gp70SU. Both antisera showed robust immunostaining of 22Rv1 cells and XMRV-transfected cells, but did not stain a number of uninfected cell lines. We tested 161 prostate tumor-derived DNA samples by real-time PCR and 596 prostate tumor tissue specimens by IHC. Our tumor tissues were enriched for cases with high Gleason score (e.g., greater than or equal to 7) and included a number of metastatic lesions. We also tested 452 prostate tissues containing a variety of benign pathologies by IHC. We did not detect XMRV in any of the samples tested. The findings suggest an extremely low prevalence, or absence, of XMRV in benign and malignant prostate tissues in men living in North America.